【作者】 赵岽琬；

【导师】 吴琼；

【作者基本信息】 哈尔滨工业大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 印记基因是基因组中一小部分亲本来源特异性沉默的基因。目前,已经在小鼠中鉴定出的印记基因数量超过100个,其中多数基因是母本表达,少数为父本表达。2009年Kobayashi等利用孤雌生殖胚胎鉴定出第一个小鼠1号染色体上的父本表达印记基因Zdbf2,本课题根据印记基因通常成簇分布于染色体上这一特点,在Zdbf2基因邻近区域检索侯选印记的EST,并对其中的EST——CJ051953进行了分离、印记鉴定及表达分析。在印记鉴定中,以C57BL/6J与ICR的杂交品系作为实验材料,采用基于SNP标记的RT-PCR直接测序法,直接分析了双亲等位基因的表达情况。印记分析结果表明CJ051953为父本优先表达的印记基因,其印记具有组织及发育阶段特异性,命名为Peg14 (paternal expression gene 14)。全胚胎原位杂交结果表明,较强的Peg14信号出现在小鼠E9.5天的菱脑、面部间充质、神经管及E11.5天的端脑、中脑和菱脑等组织。E12.5天组织切片原位杂交的结果显示,该基因在前脑、中脑、后脑和垂体中的信号较强,在脊髓及体节中相对较弱。此外,我们进一步利用全胚胎半定量RT-PCR法对该基因在胚胎发育中后期的表达情况进行了分析,结果显示,Peg14基因在发育的中后期持续性表达,其中,E9.5天表达水平最低,E11.5天表达量达到最高值。这种在脑部集中的表达模式,以及在脑中的印记状态表明,Peg14基因可能在小鼠胚胎神经系统的发育中发挥重要作用。更多还原

【Abstract】 Imprinted genes are a small proportion of genes on the genome showing parent-of-origin-specific silencing. Up to now, more than 100 imprinted genes have been identified in mouse. Most of them are expressed from the maternal allele and the others from the paternal allele. The first paternally expressed imprinted gene Zdbf2 on mouse chromosome 1 was identified by Kobayashi et al using parthenogenetic embryos in 2009. In our research, EST of candidate imprinted genes was searched in the area closed to Zdbf2 according to the characteristic feature that imprinted genes are generally physically linked in clusters with other ones on chromosomes, and the isolation, identification and expression analysis of the EST——CJ051953 were investigated.Strain cross of C57BL/6J and ICR was used as experimental material to achieve imprinting identification. Expression of parental alleles were analysed using direct sequencing of RT-PCR based on SNP. The result showed that CJ051953 which named Peg14 (paternal expression gene 14) was biased in favor of the paternal allele and presented tissue-specific and developmental stage-specific expression profiles. Whole-mount in situ hybridization showed that strong signal of Peg14 appeared in the rhombencephalon, facial mesenchyme, neural tube at E9.5 and telencephalon, midbrain, rhombencephalon at E11.5. The result of in situ hybridization at E12.5 showed that the signal of Peg14 was relatively strong in the forebrain, midbrain,hindbrain and pituitary gland, while weaker in the spinal cord and somite. In addition, expression analysis was carried out by whole embryo semi-quantitative RT-PCR. The result showed that Peg14 expressed continuously in middle-late developmental stage. Peg14 exhibited the lowest expression level at E9.5 and reached the highest expression level at E11.5.This expression profile concentrated and imprinted in the brain indicate that Peg14 may have an important effect on the development of the nervous system of the mouse embryo.更多还原